NZ613022B2 - Use of acetylated tubulin as a biomarker of drug response to furazanobenzimidazoles - Google Patents

Abstract

Disclosed is the use of acetylated tubulin as a biomarker for predicting the response to a compound, preferably resistance of a disease such as cancer in a subject to said compound, wherein the compound is a furazanobenzimidazole compound of general formula (I), in particular BAL27862.

Description

Use of acetylated tubulin as a biomarker of drug response to furazanobenzimidazoles The present invention generally relates to use of acetylated tubulin as a biomarker for predicting the response of a disease, such as a neoplastic or autoimmune disease, preferably cancer, to a compound of general formula I, such as 3-(4-{1-[2-(4-amino-phenyl)oxo-ethyl]-1H-benzoimidazolyl}-furazanylamino)- propionitrile (BAL27862). In other aspects it s to methods and kits involving the use of the biomarker. Also described are s of treatment involving the use of the ker.

Microtubules are one of the components of the cell cytoskeleton and are ed of heterodimers of alpha and beta n. Agents that target microtubules are among the most effective xic chemotherapeutic agents having a broad spectrum of activity. Microtubule destabilising agents (e.g. the vinca-alkaloids such as vincristine, vinblastine and lbine) are used for example in the treatment of several types of hematologic malignancies, such as lymphoblastic leukaemia and lymphoma, as well as solid s, such as lung cancer. Microtubule stabilising agents (e.g. the taxanes such as paclitaxel, docetaxel) are used for example in the treatment of solid tumours, including breast, lung and prostate cancer.

However resistance to these known microtubule targeting agents can occur.

The ance can either be inherent or can be acquired after exposure to these agents. Such resistance therefore impacts patient survival rates, as well as choices of treatment regimes. Several potential mechanisms of resistance have been identified, and include defects in the microtubule targets, such as elevated levels of beta-tubulin subtype III and acquired ons in beta-tubulin e I that are known to reduce taxane binding. Furthermore, defects in other cell ns have been ted to be associated with resistance to certain microtubule targeting agents, such as overexpression of the efflux pump P-glycoprotein (P-gp pump, also known as multi-drug resistance protein 1 or MDR1). Such factors may then be used as biomarkers of resistance to these conventional microtubule targeting agents.

A relatively recently discovered class of microtubule destabilising agents are compounds encompassed by the a given below: wherein R3 R2 N R4 N R5 N N R represents phenyl, thienyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently ed from alkyl, halo-lower alkyl, y-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower , hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino, tuted amino wherein the two substituents on nitrogen form together with the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein nyl is optionally substituted by lower alkoxy, amino or halogen; X represents a group C=Y, wherein Y stands for oxygen or nitrogen substituted by hydroxy or lower alkoxy; R1 represents hydrogen, lower arbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 represent hydrogen; R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R4 and R5 together represent methylenedioxy; and pharmaceutically acceptable salts thereof; or wherein R represents phenyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted amino n the two substituents on nitrogen form together with the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, lower carbonyl, formyl, cyano, halogen, and nitro; and wherein two adjacent substituents are enedioxy; and wherein pyridinyl is optionally substituted by lower , amino or halogen; X represents oxygen; R1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 represent hydrogen; R4 and R5, independently of each other, represent en, lower alkyl or lower alkoxy; or R4 and R5 together represent methylenedioxy; and pharmaceutically acceptable salts thereof; and wherein the prefix lower denotes a radical having up to and ing a maximum of 7, especially up to and including a maximum of 4 carbon atoms.

These compounds are disclosed in WO2004/103994 A1, which is orated by cross-reference herein. These compounds have been shown to arrest tumour cell proliferation and induce apoptosis.

The synthesis of compounds of formula I is described in WO2004/103994 A1, in general on pages 29-35, and specifically on pages 39-55, which are incorporated herein by cross-reference. They may be prepared as sed or by an analogous method to the processes described n.

One compound falling within this class, known as BAL27862, and shown in WO2004/103994 A1 as example 58, and specifically incorporated by reference , has the structure and chemical name given below: N N N N O Chemical name: 3-(4-{1-[2-(4-Amino-phenyl)oxo-ethyl]-1H-benzoimidazol- furazanylamino)-propionitrile; or herein as Compound A Further nds exemplified in WO2004/103994 A1 as examples 50 and 79 respectively, and also specifically incorporated by cross-reference herein, have the structures and chemical names given below: N N N N O Chemical name: 2-[2-(4-Amino-furazanyl)-benzoimidazolyl](4-amino-phenyl)- ethanone; or herein as Compound B and N N N N O Chemical name: 3-(4-{1-[2-(6-Amino-pyridinyl)oxo-ethyl]-1H- midazolyl}-furazanylamino)-propionitrile; or herein as Compound C.

BAL27862 has activity across a broad panel of experimental, solid tumour xenograft models. Moreover, activity is retained even against tumour models which were selected for resistance to tional microtubule targeting agents (including the vinca-alkaloid microtubule destabilisers and the microtubule stabilisers paclitaxel and epothilone B). BAL27862 activity is not affected by over-expression of the P-gp pump in any models tested in vitro, nor in human mammary tumour xenografts.

Additionally, 62 retained its activity despite elevated levels of beta-tubulin subtype III and mutations in n subtype I.

Hence, BAL27862 activity is not affected by a number of factors that confer resistance to conventional microtubule targeting agents.

Moreover, it is known that compounds of general formula I have a different effect on the phenotype of cells compared to other microtubule targeting agents, including other microtubule ilisers. Treatment with a compound of general formula I induces a consistent microtubule ype in tumour cell lines derived from a variety of organs, for e lung, cervix and breast, as seen in Figure 1.

Staining the microtubules in these cells with an anti alpha tubulin antibody shows that rather than the mitotic spindle fibres of untreated cells, only ke structures are visible in the treated cells. This same effect is also shown using Compounds C and B in Figures 2A and 2B tively on the lung cancer cell line A549. It is however very distinct from that observed with the conventional microtubule targeting agents vinblastine, colchicine, paclitaxel and nocodazole as seen in Figures 3B, 3C, 3D and 4, respectively. The microtubules were stained with an anti-alpha tubulin antibody and the cells viewed at a 1000 x magnification (Figures 3, 4). For the cells treated with BAL27862, multiple dot-like structures are visible, whereas, in stark contrast, the other conventional drugs produce filamentous microtubule structures, or dense microtubule aggregate structures. These differences at the phenotypic level, at compound doses considered optimal in terms of antiproliferative , indicate a difference in the mode of action at the molecular level.

Furthermore, it is known that BAL27862 elicits a dominant microtubule phenotype in the presence of the other microtubule targeting agents. Treatment with vinblastine, colchicine, axel or nocodazole alone induced the microtubule ypes characteristic of these agents (Figure 5A, 5D, 5G, 6C-6F respectively).

However, combination treatment with BAL27862 for the last 4 hours resulted in disruption of these phenotypes; despite the continued presence of vinblastine, colchicine, paclitaxel or nocodazole (Figure 5B, 5E, 5H, 6G-6J respectively). In contrast, treating first with 62 and uently for 4 hours in combination with vinblastine, colchicine, paclitaxel or nocodazole had no impact on generation of the phenotype consistent with BAL27862 treatment (Figure 5C, 5F, 5I, 6K-6N respectively).

These data all demonstrate that 62 s microtubule biology in a different manner than conventional microtubule ing .

Thus, from information about conventional microtubule targeting agents, predictions cannot be made ning if, or how, particular genes are involved in the action of compounds of formula I.

An object of the present invention is to identify factors which are ated with response to compounds of formula I or pharmaceutically acceptable derivatives thereof, for example to identify factors associated with resistance to compounds of general formula I, in particular BAL27862 or pharmaceutically acceptable derivatives thereof, as d below; and/or to provide the public with a useful choice.

It has surprisingly been found that acetylated tubulin may be used as a biomarker of response to ent with a compound of general formula I or pharmaceutically able derivatives thereof, as defined below.

Accordingly, in a first aspect the ion relates to the use of acetylated tubulin as a ker for predicting the response to a compound, wherein the R3 R2 N R4 N R5 N N compound is a compound of general formula I wherein R represents phenyl, thienyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower carbonylamino, lower alkylcarbonylamino, substituted amino wherein the two substituents on nitrogen form together with the nitrogen cyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein nyl is optionally substituted by lower alkoxy, amino or halogen; X represents a group C=Y, wherein Y stands for oxygen or nitrogen substituted by hydroxy or lower alkoxy; R1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 represent hydrogen; R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R4 and R5 together represent methylenedioxy; and pharmaceutically acceptable salts, solvates, esters and amides of naturally occurring amino acids, small peptides or pegylated hydroxy acids, salts of such esters and , and polymorphs thereof; or wherein R represents phenyl or pyridinyl wherein phenyl is optionally substituted by one or two tuents independently selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, y-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower arbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted amino wherein the two substituents on nitrogen form together with the nitrogen heterocyclyl, lower arbonyl, carboxy, lower alkoxycarbonyl, formyl, cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein pyridinyl is optionally substituted by lower alkoxy, amino or halogen; X represents oxygen; R1 represents en, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 represent hydrogen; R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower or R4 and R5 er represent methylenedioxy; and pharmaceutically able salts, solvates, esters and amides of naturally occurring amino acids, small peptides or pegylated hydroxy acids, salts of such esters and amides, and polymorphs thereof, and wherein the prefix lower denotes a radical having up to and including a maximum of 7 carbon atoms and wherein the response is of a disease in a subject and the biomarker acetylated tubulin is measured ex vivo in a sample or samples taken from the human or animal body.

In a second aspect, the invention provides a method for predicting in a subject suffering from a cancer the response of that cancer to a compound of general formula I or a pharmaceutically acceptable salt, solvate, ester or amide of a naturally occurring amino acid, small e or pegylated hydroxy acid, or a salt of such ester or amide, or polymorph thereof as defined in the first aspect of the invention, sing the steps of: a1) measuring ex vivo a level of acetylated tubulin in a sample pre-obtained from tumour tissue or circulating tumour cells of the subject suffering from the disease, which is a cancer, to obtain a value or values representing this level; or a2) measuring ex vivo a level of ated tubulin in a sample pre-obtained from the subject suffering from the disease, after initiation of treatment with the nd of general formula I, to obtain a value or values representing this level; b1) comparing the value or values from step a1) to a standard value or set of standard values from subjects with the same cancer type, n a higher level of acetylated tubulin in the sample from the subject relative to the standard value or set of standard values predicts resistance; or b2) comparing the value or values from step a2) to the pre-treatment initiation level of that subject, wherein a higher level of ated tubulin in the sample after treatment initiation relative to the pre-treatment initiation level ts acquired resistance.

In a third aspect, the invention relates to the use of a compound of general formula I or of a pharmaceutically acceptable salt, solvate, ester or amide of a naturally occurring amino acid, small peptide or pegylated hydroxy acid, or a salt of such ester or amide, or polymorph thereof as defined in a first aspect of the invention, for the ation of a ceutical composition for treating a cancer in a human t in need thereof, wherein the human subject is selected for treatment with the compound of general formula I or with the salt, solvate, ester or amide of a naturally occurring amino acid, small peptide or pegylated hydroxy acid, or salt of such ester or amide, or polymorph thereof as defined in a first aspect of the invention, if the level of ated tubulin, measured ex vivo in a sample taken from the human subject, is not higher than a standard value or set of standard values from subjects with the same tumour histotype or from normal cells, tissue or body fluid, wherein a higher value of acetylated tubulin in the sample ed from the human subject relative to the standard value or set of standard values is predictive of resistance to the compound of formula (I), or to the pharmaceutically acceptable salt, solvate, ester or amide of a naturally occurring amino acid, small peptide or pegylated hydroxy acid, or to the salt of such ester or amide, or to the polymorph thereof.

In a fourth aspect, the ion es a kit when used for predicting the response to a compound of general formula I or a pharmaceutically acceptable salt, solvate, ester or amide of a naturally occurring amino acid, small peptide or pegylated hydroxy acid, or a salt of such ester or amide, or polymorph thereof, as defined in a first aspect of the ion, comprising reagents necessary for measuring a level of acetylated tubulin in a sample taken from a t with a cancer, and further comprising a comparator module which comprises a standard value or set of standard values of a level of acetylated tubulin taken from samples of tumour tissue or circulating tumour cells of subjects with a cancer of the same histotype to which the level of acetylated tubulin in the sample is compared, wherein said reagents comprise a capture reagent sing a detector for acetylated tubulin as d in a first aspect of the invention and a detector reagent, and n the kit comprises a compound of the following formula or a pharmaceutically acceptable salt thereof, N N N N O Certain statements that appear below are broader than what appears in the statements of the invention above. These statements are provided in the interests of providing the reader with a better tanding of the invention and its practice. The reader is directed to the accompanying claim set which s the scope of the invention.

In one preferred embodiment of the invention, relatively high acetylated tubulin levels in a tumour sample are ated with resistance to BAL27862, as described below.

Tubulin is subjected to a variety of post-translational cations, including detyrosination/tyrosination, acetylation, glutamylation, polyglycylation, phosphorylation of serine residues and phosphorylation of tyrosine residues, making it one of the most modified proteins known.

The acetylation and deacetylation of lysines in n is known to occur, although the exact function of these changes has yet to be elucidated. The best characterised site of acetylation is on the alpha tubulin lysine 40, however additional acetylation sites have been identified on both alpha and beta tubulin. To date two ubule deacetylases have been identified: histone deacetylase 6 (HDAC6) and Sirtuin T2 (SirT2). Very recently AT1 and the Elp3 subunit of the tor complex were identified as lysine 40 tubulin acetyltransferases. Furthermore, the protein known as San edly has lysine 252 beta-tubulin acetyltransferase activity. (A novel acetylation of beta-tubulin by San modulates microtubule polymerization via down regulating tubulin oration, Chih-Wen Chu et al., Mol Biol Cell. 2010, Dec 22.) Acetylated tubulin is also known as acetylated-tubulin, acetylated microtubule(s), or acetyl tubulin, and the term acetylated n shall be used herein to also encompass these synonyms. The designation acetylated n, shall also ass forms wherein other post-translational modifications may additionally be present. The alpha and beta tubulin which form the basis for the acetylated tubulin are known to exist in multiple variants, es and isoforms, as well as there being multiple alpha and beta tubulin genes which give rise to these. Preferably the tubulin which is acetylated relates to human variants, subtypes and isoforms of alpha and beta tubulin, more preferably to human variants, subtypes and isoforms of alpha tubulin. Subtypes of alpha n include, but are not limited to, tubulin alpha 1A, n alpha 1B, tubulin alpha 1C, tubulin alpha 2, n alpha 3C/D, tubulin alpha 4A, and tubulin alpha-8 chain isoform 1. The subtypes tubulin alpha 1A, tubulin alpha 1B, tubulin alpha 1C, n alpha 2, tubulin alpha 3C/D and tubulin alpha 4A possess a lysine 40 and are thus a preferred subset of alpha tubulins ing to the invention. Subtypes of beta tubulin include, but are not limited to, tubulin beta chain, n beta-1 chain and tubulin beta-3 chain isoform 1. The protein sequences of the alpha tubulin subtypes are accessible via the following National Center for Biotechnology Information (NCBI) nce numbers NP_006000, NP_006073, NP_116093, ABD72607, NP_005992, NP_005991 and NP_061816, and the beta tubulin subtypes are accessible via NP_821133, NP_110400 and NP_006077 respectively. In an alternatively preferred embodiment the tubulin which is acetylated is selected from the group consisting of tubulin alpha-1C (NP_116093.1), tubulin alpha 3C/D (NP_005992.1), tubulin alpha-4A (NP_005991.1) tubulin alpha-8 chain isoform 1 (NP_061816.1), tubulin beta chain (NP_821133.1) and tubulin beta-3 chain isoform 1 (NP_006077.2). The polypeptide sequences of these are also listed in Figures 9-14 as SEQ ID NO. 1-6, respectively. Particularly preferably the tubulin which is acetylated is selected from the group consisting of n alpha-1C, tubulin alpha 3C/D, tubulin alpha-4A and tubulin alpha-8 chain isoform 1. More particularly preferably the tubulin which is acetylated is selected from the group consisting of tubulin alpha-1C, tubulin alpha 3C/D and tubulin alpha-4A.

One embodiment s to use of acetylated n as a biomarker for predicting the se to a compound, wherein the compound is a compound of l formula I, R3 R2 N R4 N R5 N N wherein R ents phenyl, thienyl or pyridinyl wherein phenyl is ally tuted by one or two substituents independently selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted amino wherein the two substituents on nitrogen form together with the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein pyridinyl is ally substituted by lower alkoxy, amino or halogen; X represents a group C=Y, wherein Y stands for oxygen or nitrogen substituted by hydroxy or lower alkoxy; R1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 represent hydrogen; R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R4 and R5 er represent methylenedioxy; and pharmaceutically acceptable derivatives thereof, or wherein R represents phenyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently selected from alkyl, ower alkyl, hydroxy-lower alkyl, lower -lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower -lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower arbonylamino, tuted amino wherein the two substituents on nitrogen form together with the nitrogen heterocyclyl, lower alkylcarbonyl, y, lower alkoxycarbonyl, , cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein pyridinyl is optionally substituted by lower alkoxy, amino or halogen; X represents oxygen; R1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 represent hydrogen; R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R4 and R5 together represent methylenedioxy; and pharmaceutically acceptable tives thereof; and wherein the prefix lower denotes a radical having up to and including a maximum of 7, especially up to and including a maximum of 4 carbon atoms.

Preferably the response may be of a disease in a subject. Also preferably the response may be to treatment, i.e. to treatment with the compound of general formula I or pharmaceutically acceptable derivatives thereof.

The biomarker acetylated tubulin is measured ex vivo in a sample or samples taken from the human or animal body, preferably taken from the human body.

In a preferred embodiment, described is use of acetylated tubulin as a biomarker for ting the resistance of a disease in a subject to a compound of general formula I or pharmaceutically acceptable derivatives thereof as defined above.

Preferably the pharmaceutically acceptable tive is selected from the group consisting of a salt, solvate, pro-drug, salt of a ug, rph and isomer of a compound of general formula I as defined above. Pro-drugs are preferably ester and amides of naturally ing amino acids, small peptides or pegylated hydroxy acids. More preferably, the pro-drug is an amide formed from an amino group t within the R group of the compound of general formula I and the carboxy group of glycine, alanine or lysine.

Particularly preferably the compound is N N N N O or a pharmaceutically acceptable salt thereof, preferably a hydrochloride salt, most preferably a dihydrochloride salt thereof. r embodiment relates to a method for predicting the response of a disease in a subject to a compound of general formula I or pharmaceutically acceptable derivatives thereof as defined above, comprising the steps of: a) measuring a level of acetylated tubulin in a sample pre-obtained from the subject to obtain a value or values representing this level; and b) comparing the value or values from step a) to a rd value or set of standard values or to pre-treatment initiation levels.

Further preferably the response which is to be predicted is resistance.

The measuring of a level or levels of acetylated n is performed ex vivo in a sample or samples pre-obtained from the subject. Pre-obtained refers to the fact that the sample is obtained before it is subjected to any method involving ing the level of the biomarker, and pre-obtained is not to be understood as in relation to treatment.

In a preferred embodiment, a higher level of acetylated tubulin in the sample from the t relative to the standard value or set of standard values or pretreatment initiation levels predicts resistance.

Also preferably, the e is a stic or autoimmune disease. More preferably the disease is cancer. ally preferably, the cancer is selected from the group consisting of breast cancer, prostate cancer, cervical , ovarian cancer, gastric , colorectal cancer (i.e. including colon cancer and rectal cancer), atic cancer, liver cancer, brain cancer, neuroendocrine cancer, lung cancer, kidney cancer, hematological malignancies, melanoma, T-cell leukemia, and sarcomas. More especially preferably the cancer is selected from the group consisting of breast cancer, cervical cancer, ovarian cancer, T-cell leukemia and lung cancer. In an especially preferred embodiment the cancer is selected from the group consisting of lung cancer, ovarian cancer and T-cell leukemia.

Also described is a method of ng a neoplastic or autoimmune disease, preferably cancer, in a subject in need f, comprising measuring a level of acetylated tubulin in a sample from the subject to obtain a value or values representing this level, and treating the subject with a compound of general formula I or a pharmaceutically acceptable derivative thereof as defined above, if the level of acetylated tubulin in said sample is not higher than a standard value or set of standard values or pre-treatment initiation levels.

Also bed is ated tubulin for use in the treatment of a neoplastic or autoimmune e, preferably cancer, comprising measuring a level of acetylated tubulin in a sample from the subject to obtain a value or values enting this level, and treating the subject with a nd of general formula I or a pharmaceutically acceptable derivative thereof as defined above, if the level of acetylated tubulin is not higher than a standard value or set of standard values or pre-treatment initiation levels.

The measuring of a level of acetylated tubulin is performed ex-vivo in a sample pre-obtained from the t.

Also described is a method of treating a neoplastic or autoimmune disease, preferably cancer, by first decreasing the level of ated tubulin in a subject that has a sample with a higher level of acetylated tubulin compared to a standard level or set of standard levels or pre-treatment initiation levels, then treating the subject with a compound of general formula I or a pharmaceutically acceptable derivative thereof as defined above.

Also described is a kit for predicting the response to a compound of general formula I or a pharmaceutically able derivative f, as defined above, comprising reagents necessary for measuring a level of acetylated tubulin in a . More preferably the kit also ses a comparator module which comprises a standard value or set of standard values to which the level of acetylated tubulin in the sample is compared.

Furthermore preferably the kit comprises a compound of general formula I or a pharmaceutically acceptable derivative thereof as defined above. In an especially preferred embodiment the kit comprises a compound of the following a or a ceutically acceptable salt thereof N N N N O Chemical name: S-2,6-Diamino-hexanoic acid [4-(2-{2-[4-(2-cyanoethylamino )-furazanyl]-benzoimidazolyl}-acetyl)-phenyl]-amide In a particularly preferred embodiment the pharmaceutically acceptable salt is a dihydrochloride salt.

Also described is a device for predicting the response to a compound of general formula I or a ceutically acceptable derivative thereof as defined above, comprising reagents ary for measuring a level of acetylated tubulin in a sample and a comparator module comprising a standard value or set of standard values to which the level of acetylated tubulin in the sample is compared.

In a preferred embodiment, the reagents in the kit or device se a capture t sing a detector for acetylated tubulin, and a detector reagent.

Especially preferably the capture reagent is an antibody. Also preferably, the disease is predicted to be resistant to treatment with said compound when acetylated tubulin is higher relative to a standard value or set of rd values or pre-treatment initiation levels. In a preferred embodiment, the comparator module is included in instructions for use of the kit. In another preferred embodiment the comparator module is in the form of a display device.

Embodiments of the present invention will now be bed by way of example with reference to the accompanying figures. The invention however is not to be understood as d to these ments.

Brief Description of the Figures Figure 1: Shows the treatment of human tumour cell lines from different histotypes with 50 nM BAL27862. The microtubules of mitotic or G2/M arrested cells were stained after 24 hours treatment with 50 nM BAL27862 or vehicle control.

Fig. 1A and 1B: A549 NSCLC cells; Fig. 1C and 1D: HeLa cervical cancer cells; Fig. 1E and 1F: SKBR3 breast cancer cells Vehicle control treatment: Figures 1A, 1C & 1E, BAL27862 treatment: Figures 1B, 1D & 1F.

Figure 2: Shows the treatment of A549 NSCLC cells with the Compounds B and C. The microtubules of mitotic or G2/M ed A549 NSCLC cells were stained after 24 hours treatment with 80 nM or 20 nM of Compounds B and C, respectively.

The white scale bar ents 10 micrometres.

Fig. 2A: treatment with 20 nM Compound C Fig. 2B: treatment with 80 nM nd B Figure 3: Shows a comparison of treatment of cells with BAL27862 compared to conventional microtubule targeting agents. Microtubules of mitotic or G2/M arrested A549 NSCLC cells were stained after 24 hours of treatment with 50 nM of A: BAL27862; B: stine; C: colchicine; D: paclitaxel. Stacks of images taken every 1 µm were processed by using ImageJ software.

Figure 4: Shows a comparison of treatment of A549 NSCLC cells with BAL27862 ed to nocodazole. Microtubules of c or G2/M ed cells were stained after 24 h of treatment with various concentrations of nocodazole (B, C & D) and BAL27862 (E, F & G). A: control, B: Nocodazole 50 nM, C: Nocodazole 100 nM, D: Nocodazole 200 nM, E: BAL27862 20 nM; F: BAL27862 30 nM and G: BAL27862 50 nM. The white scale bar represents 10 micrometres. Representative images of the microtubule phenotypes observed are shown.

Figure 5: Shows a combination of treatment with BAL27862 and conventional microtubule-targeting agents. Microtubules of c or G2/M arrested A549 NSCLC cells were stained after treatment for the times indicated below. 50 nM BAL27862, 50 nM vinblastine, 50 nM colchicine and 25 nM paclitaxel were used. The white scale bar represents 10 micrometres.

Fig. 5A: 24 hours vinblastine treatment; Fig. 5B: 24 hours vinblastine treatment with the final 4 hours including BAL27862; Fig. 5C: 24 hours BAL27862 treatment with the final 4 hours including vinblastine.

Fig. 5D: 24 hours colchicine treatment; Fig. 5E: 24 hours colchicine treatment with the final 4 hours including Fig. 5F: 24 hours BAL27862 treatment with the final 4 hours including cine.

Fig. 5G: 24 hours paclitaxel treatment; Fig. 5H: 24 hours axel treatment with the final 4 hours including Fig. 5I: 24 hours BAL27862 treatment with the final 4 hours including paclitaxel.

Figure 6: Shows a combination of treatment with BAL27862 and nocodazole. Microtubules of mitotic or G2/M arrested A549 NSCLC cells were stained after treatment for the times indicated below. 25 nM BAL27862 and nocodazole at the concentrations indicated below were used. The white scale bar represents 10 micrometers.

Fig. 6A: 24 hours control treatment; Fig. 6B: 24 hours of 25 nM 62 treatment; Fig. 6C: 24 hours of 50 nM nocodazole treatment Fig. 6D: 24 hours of 100 nM nocodazole treatment Fig. 6E: 24 hours of 150 nM nocodazole treatment Fig. 6F: 24 hours of 200 nM nocodazole treatment Fig. 6G: 24 hours of 50 nM zole treatment with the final 4 hours ing 25 nM BAL27862; Fig. 6H: 24 hours of 100 nM nocodazole treatment with the final 4 hours ing 25 nM BAL27862; Fig. 6I: 24 hours of 150 nM nocodazole treatment with the final 4 hours including 25 nM BAL27862; Fig. 6J: 24 hours of 200 nM nocodazole treatment with the final 4 hours including 25 nM BAL27862; Fig. 6K: 24 hours of 25 nM BAL27862 treatment with the final 4 hours including 50 nM nocodazole; Fig. 6L: 24 hours of 25 nM 62 treatment with the final 4 hours including 100 nM nocodazole; Fig. 6M: 24 hours of 25 nM BAL27862 treatment with the final 4 hours including 150 nM nocodazole; Fig. 6N: 24 hours of 25 nM BAL27862 treatment with the final 4 hours including 200 nM nocodazole.

Figure 7: Shows tumour cell lines which were selected for resistance to BAL27862 through in vitro cultivation in the presence of the compound. Based on IC 50 (for proliferation: A549, SKOV3, H460) or EC50 (for cell death: Jurkat) inations, BAL27862 resistance factors versus parental lines were: A549 (3.0 fold); SKOV3 (7.6 fold – resistant 1 line); Jurkat (22.5 fold), H460 (5.3 fold) (see Table 1). Whole cell protein extracts were prepared from parental and resistant lines and analysed by immunoblotting for ated tubulin expression. Actin levels were included as a loading control.

Figure 8: Shows that increased acetylated tubulin protein levels are maintained in SKOV3 tumour lines during resistance development. SKOV3 tumour cell lines were ed for resistance to BAL27862 through in vitro cultivation in the presence of 62 for increasing time periods. Based on IC50 determinations, BAL27862 resistance factors versus parental lines were: SKOV3 resistant 1 (7.6 fold), SKOV3 resistant 2 (11.6 fold) (see Table 1). Whole cell protein extracts were prepared from parental and resistant lines and ed by immunoblotting for acetylated tubulin sion. Actin levels act as a loading control.

Figure 9: Shows the protein sequence of tubulin alpha-1C chain [Homo sapiens] (SEQ. ID. No. 1) Figure 10: Shows the n sequence of tubulin alpha-3C/D chain [Homo sapiens] (SEQ ID No. 2) Figure 11: Shows the protein sequence of tubulin alpha-4A chain [Homo sapiens] (SEQ. ID. NO. 3) Figure 12: Shows the protein sequence of tubulin alpha-8 chain isoform 1 [Homo sapiens] (SEQ. ID. NO. 4) Figure 13: Shows the protein sequence of tubulin beta chain [Homo sapiens] (SEQ. ID. No. 5) Figure 14: Shows the protein sequence of n beta-3 chain isoform 1 [Homo sapiens] (SEQ. ID. NO. 6) Detailed Description Compounds of formula I The compounds described herein are represented by general formula I: wherein R3 R2 N R4 N R5 N N R represents , thienyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, y, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower arbonylamino, substituted amino wherein the two substituents on nitrogen form together with the en heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein pyridinyl is optionally substituted by lower alkoxy, amino or halogen; X represents a group C=Y, wherein Y stands for oxygen or nitrogen substituted by hydroxy or lower ; R1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 represent hydrogen; R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R4 and R5 together represent methylenedioxy; and pharmaceutically acceptable derivatives thereof, or wherein R represents phenyl or pyridinyl wherein phenyl is optionally substituted by one or two tuents independently selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, , hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted amino wherein the two tuents on en form together with the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, formyl, cyano, halogen, and nitro; and n two adjacent substituents are methylenedioxy; and wherein pyridinyl is optionally substituted by lower , amino or halogen; X represents oxygen; R1 represents hydrogen, lower arbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 ent hydrogen; R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R4 and R5 together represent methylenedioxy; and pharmaceutically acceptable derivatives thereof; and wherein the prefix lower denotes a radical having up to and including a maximum of 7, especially up to and including a maximum of 4 carbon atoms.

Heterocyclyl designates preferably a saturated, partially saturated or unsaturated, mono- or bicyclic ring containing 4-10 atoms comprising one, two or three heteroatoms selected from nitrogen, oxygen and sulfur, which may, unless otherwise specified, be carbon or nitrogen linked, wherein a ring nitrogen atom may optionally be substituted by a group selected from lower alkyl, amino-lower alkyl, aryl, aryl-lower alkyl and acyl, and a ring carbon atom may be substituted by lower alkyl, lower alkyl, aryl, aryl-lower alkyl, aryl, lower alkoxy, hydroxy or oxo.

Examples of heterocyclyl are pyrrolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, morpholinyl, piperazinyl, dioxolanyl and ydropyranyl.

Acyl designates, for example, alkylcarbonyl, cyclohexylcarbonyl, arylcarbonyl, aryl-lower alkylcarbonyl, or arylcarbonyl. Lower acyl is ably lower alkylcarbonyl, in particular propionyl or acetyl.

Preferably, the compound of l formula I is defined as wherein R1 is selected from the group consisting of hydrogen, acetyl, CH2CH 2CN and CH 2CH 2CH 2OH.

In one preferred embodiment, the compound of general formula I is selected from the group consisting of: 4-(1-Phenacyl-1H-benzimidazolyl)-furazanylamine, 4-[1-(4-Bromophenacyl)-1H-benzimidazolyl]-furazanylamine oxime, 1-(4-Chlorophenacyl)-1H-benzimidazolyl]-furazanyl}-acetamide, 4-[1-(4-Chlorophenacyl)-1H-benzimidazolyl]-furazanyl-N-(2-cyanoethyl)-amine 4-[1-(4-Chlorophenacyl)-1H-benzimidazolyl]-furazanyl-N-(3-hydroxypropyl)- amine, 4-[1-(3-Aminochlorophenacyl)-1H-benzimidazolyl]-furazanylamine 4-[1-(3-Methoxymethoxymethoxy-phenacyl)-1H-benzimidazolyl]-furazan ylamine, and pharmaceutically able derivatives thereof.

In another preferred embodiment, the compound of general formula I is selected from the group consisting of: N N wherein R, Y and R1 are d as follows : R Y R1 O H NOH H NOMe H O H NOH H NOH H NOMe H O H NOH H NOMe H O H NOH H NOMe H O H NOMe H O H Cl Cl O H NOH H NOMe H O H NOMe H NOMe H O H Et2N O Ac O H O H O H O CH2CH2CN O CN O H O H O CH2CH2CH2OH O H O CH2CH2CN O H O CN O CH2CH2CN O CH2CH2CN O H AcNH O H O H AcHN O H O H O H O H O CN O H O H O H O H O H O H O H O H O H NH N O CH2CH2CN NH N O H O O O H O O O CH2CH2CN MeO N or pharmaceutically acceptable tives thereof.

In yet another preferred embodiment, the compound of general formula I is ed from the group consisting of: 4-(1-Phenoxymethyl-1H-benzimidazolyl)-furazanylamine, 4-[1-(4-Fluorophenoxymethyl)-1H-benzimidazolyl]-furazanylamine, 4-[1-(3,4-Dimethylphenoxymethyl)-1H-benzimidazolyl]-furazanyl-N-(2- cyanoethyl)-amine and compounds represented by the formula: N N N N O wherein R and R1 are as defined below R R1 CH2CH2CN CH2CH2CN CH2CH2CN CH2CH2CH2OH Cl N NH N and pharmaceutically acceptable derivatives f.

In still yet another preferred embodiment the compound of general formula I R4 N N N N O wherein R, R4 and R5 are as defined below R R4 R5 Me Me Me Me Me Me Me Me Me Me OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe or pharmaceutically acceptable derivatives thereof.

More ably, the compound is a compound of general formula I R3 R2 N R4 N R5 N N wherein R represents phenyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently selected from lower alkyl, lower , amino, acetylamino, halogen and nitro; and wherein nyl is optionally substituted by amino or halogen; X represents a group C=O; R1 represents hydrogen or cyano-lower alkyl; R2, R3, R4, R5 and R6 represent hydrogen; and pharmaceutically acceptable derivatives thereof, and n the prefix lower denotes a l having up to and including a maximum of 7, especially up to and including a maximum of 4 carbon atoms.

Especially preferably, the compound is represented by the following formula N N wherein R, Y and R1 are defined as follows : R Y R1 O H O CH2CH2CN O H NH N O CH2CH2CN NH N or pharmaceutically acceptable derivatives thereof.

More ally preferably, the compound is represented by the following formula N N wherein R, Y and R1 are defined as follows: R Y R1 O CH2CH2CN O H O CH2CH2CN NH N or pharmaceutically acceptable derivatives thereof.

Particularly preferably, the compound is N N N N O or pharmaceutically acceptable derivatives thereof.

The term derivative or derivatives in the phrase “pharmaceutically acceptable derivative” or “pharmaceutically acceptable derivatives” of compounds of general formula I relates to salts, solvates and complexes thereof and to es and complexes of salts f, as well as to pro-drugs, polymorphs, and isomers thereof (including optical, geometric and tautomeric isomers) and also salts of pro- drugs thereof. In a more red embodiment, it relates to salts and pro-drugs, as well as to salts of pro-drugs thereof.

Salts are ably acid addition salts. Salts are formed, preferably with organic or inorganic acids, from compounds of formula (I) with a basic nitrogen atom, especially the pharmaceutically acceptable salts. Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid.

Suitable organic acids are, for e, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, ic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, c acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, maleic acid, cyclohexanecarboxylic acid, tanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1,5- naphthalene-disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid, methylsulfuric acid, ulfuric acid, dodecylsulfuric acid, ohexylsulfamic acid, N-methyl-, N- ethyl- or N-propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid.

The compound described herein may be administered in the form of a prodrug which is broken down in the human or animal body to give a compound of the formula I. Examples of pro-drugs include in vivo hydrolysable esters and amides of a compound of the formula I. Particular pro-drugs considered are ester and amides of naturally occurring amino acids and ester or amides of small peptides, in particular small peptides consisting of up to five, preferably two or three amino acids as well as esters and amides of pegylated y acids, preferably hydroxy acetic acid and lactic acid. Pro-drug esters are formed from the acid on of the amino acid or the C al of the peptide and suitable y group(s) in the compound of a I.

Pro-drug amides are formed from the amino function of the amino acid or the N terminal of the peptide and suitable carboxy group(s) in the compound of formula I, or from the acid function of the amino acid or the C terminal of the peptide and suitable amino group(s) in the compound of formula I. Particularly preferably the pro-drug amides are formed from the amino group(s) present within the R group of formula I.

More preferably, the pro-drug is formed by the addition of glycine, alanine or lysine to the nd of formula I.

Even more preferably the nd of general formula I is in the form of a pro-drug selected from the compounds of formulae: NH N N N N N N O N N O N N O O O H N O H N NH2 , NH H2N 2 , , NH N N O N N N N N N O N N O O N N O N H N O N NH2 H H , NH H2N 2 , , H N N 2 H N N 2 N N O N N N N N O N N O O O O O H N O N NH H 2 2 NH H2N , 2 and .

In an especially preferred embodiment the compound of general formula I is in the form of a pro-drug which has the following formula N N N N O In a most especially preferred embodiment the compound is a salt, preferably a hydrochloride salt, most preferably a dihydrochloride salt, of a compound of the ing formula N N N N O The pharmaceutically active lite in vivo in this case is BAL27862.

These pro-drugs may be ed by processes that are known per se, in particular, a process, n a compound of formula (II) N N N N O (II) z 2 wherein R1 is defined as for formula (I) and Z is CH or N, or a derivative of such a compound comprising functional groups in protected form, or a salt thereof is (1) ed with an amino acid of formula (III) R11 (III) wherein R10 is selected from hydrogen (Gly); methyl (Ala) and protected aminobutyl (Lys) and R11 is a suitable amino protecting group, and (2) any protecting groups in a protected derivative of the resulting compound are removed to yield a pro-drug as shown above, and, if so desired, (3) said pro-drug is converted into a salt by treatment with an acid, or a salt of a compound of formula (II) is ted into the corresponding free compound of formula (II) or into another salt, and/or a mixture of ic product compounds is separated into the individual s.

Acylation of a compound of formula (II) with an amino acid of formula (III) is performed in a manner known per se, usually in the presence of a suitable polar or r aprotic solvent, with cooling or heating as required, for example in a temperature range from approximately minus 80°C to approximately plus 150°C, more preferably from minus 30°C to plus 120°C, especially in a range from approximately around 0°C to the reflux temperature of the used solvent. ally a suitable base is added, in particularly an aromatic base like pyridine or collidine or a tertiary amine base such as triethylamine or diisopropylethylamine, or an inorganic basic salt, e.g. potassium or sodium carbonate.

Acylation may be accomplished under conditions used for amide formation known per se in peptide chemistry, e.g. with activating agents for the carboxy group, such as carbodiimides like iethyl-, N,N’-dipropyl-, N,N’-diisopropyl-, N,N’- dicyclohexylcarbodiimide and N-(3-dimethylaminoisopropyl)-N’-ethylcarbodiimide- hydrochloride (EDC), or with agents such as 1-hydroxybenzotriazole (HOBt), benzotriazolyloxytris(dimethylamino)-phosphonium hexafluorophosphate (BOP), O-(7-aza-benzotriazolyl)-N,N,N’,N’-tetramethyl-uronium hexafluorophosphate (HATU), 2-(2-oxo(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU), optionally in the ce of suitable bases, catalysts or co-reagents. The carboxy group may also be activated as acyl halogenide, preferably as acyl chloride, e.g. by reaction with thionylchloride or oxalylchloride, or as symmetrical or unsymmetrical anhydride, e.g. by on with halogeno formates like ethyl chloroformate, optionally in the presence of suitable bases, catalysts or co-reagents.

If one or more other functional groups, for example carboxy, y or amino, are or need to be protected in a compound of formula (II) or (III), because they should not take part in the reaction, these are such protecting groups as are y d in the synthesis of amides like, in particular peptide compounds, cephalosporins, penicillins, nucleic acid derivatives and sugars, which are known to the skilled persons. le protecting groups for amino groups are for e tbutyl ate, benzyl carbamate or 9-fluorenylmethyl carbamate.

The protecting groups may already be present in precursors and should protect the functional groups concerned against ed secondary reactions, such as alkylations, acylations, etherifications, esterifications, oxidations, solvolysis, and similar reactions. It is a characteristic of ting groups that they lend themselves readily, i.e. without undesired secondary ons, to removal, typically by solvolysis, reduction, photolysis or also by enzyme activity, for example under conditions analogous to physiological conditions, and that they are not present in the end products. The list knows, or can easily establish, which protecting groups are suitable with the reactions mentioned hereinabove and hereinafter.

The protection of such functional groups by such protecting groups, the protecting groups themselves, and their removal reactions are described for example in standard reference books for peptide synthesis and in special books on protective groups such as J. F. W. , "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973, in "Methoden der schen Chemie" (Methods of organic chemistry), Houben-Weyl, 4th edition, Volume 15/I, Georg Thieme , Stuttgart 1974, and in T. W. Greene, G. M. Wuts "Protective Groups in Organic Synthesis", Wiley, New York, 2006.

Disease The compounds of general formula I have been shown to arrest cell proliferation and induce cell death, for example by apoptosis.

Dysregulation of cell proliferation, or lack of appropriate cell death, has wide ranging clinical implications. A number of diseases associated with such dysregulation involve hyperproliferation, inflammation, tissue remodeling and .

Familiar indications in this category include cancers, restenosis, neointimal hyperplasia, angiogenesis, endometriosis, lymphoproliferative disorders, transplantation d pathologies (graft rejection), polyposis, loss of neural function in the case of tissue remodeling and the like.

Cancer is associated with abnormal cell proliferation and cell death rates. As apoptosis is inhibited or delayed in most types of proliferative, stic diseases, induction of apoptosis is an option for treatment of cancer, ally in cancer types which show resistance to c chemotherapy, radiation and immunotherapy (Apoptosis and Cancer Chemotherapy, Hickman and Dive, eds., Blackwell Publishing, 1999). Also in autoimmune and lantation related diseases and pathologies compounds inducing apoptosis may be used to restore normal cell death processes and therefore can eradicate the symptoms and might cure the diseases. r ations of compounds inducing apoptosis may be in restenosis, i.e. accumulation of vascular smooth muscle cells in the walls of arteries, and in persistent infections caused by a failure to eradicate bacteria-and virus-infected cells.

Furthermore, apoptosis can be induced or reestablished in lial cells, in endothelial cells, in muscle cells, and in others which have lost t with extracellular matrix.

A compound according to general formula I or pharmaceutically acceptable derivatives thereof may be used for the prophylactic or especially therapeutic treatment of the human or animal body, in particular for treating a neoplastic disease, autoimmune disease, transplantation related pathology and/or degenerative disease.

Examples of such stic diseases include, but are not limited to, epithelial neoplasms, squamous cell neoplasms, basal cell neoplasms, tional cell omas and carcinomas, as und adenocarcinomas, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous neoplasms, ducal-, lobular and medullary sms, acinar cell neoplasms, complex epithelial neoplasms, specialized l neoplasms, paragangliomas and glomus tumors, naevi and melanomas, soft tissue tumors and sarcomas, atous neoplasms, myxomatous sms, lipomatous neoplasms, myomatous neoplasms, complex mixed and stromal sms, fibroepithelial neoplasms, synovial like neoplasms, mesothelial neoplasms, germ cell neoplasms, trophoblastic neoplasms, mesonephromas, blood vessel tumors, lymphatic vessel tumors, osseous and chondromatous neoplasms, giant cell tumors, miscellaneous bone tumors, odontogenic tumors, gliomas, neuroepitheliomatous neoplasms, meningiomas, nerve sheath tumors, granular cell tumors and alveolar soft part sarcomas, n's and non-Hodgkin's lymphomas, other lymphoreticular sms, plasma cell tumors, mast cell tumors, immunoproliferative diseases, leukemias, miscellaneous myeloproliferative disorders, lymphoproliferative disorders and myelodysplastic syndromes.

The compounds of general formula I or pharmaceutically acceptable derivatives thereof may be used to treat autoimmune diseases. Examples of such autoimmune diseases include, but are not limited to, systemic, discoid or subacute cutaneous lupus matosus, rheumatoid arthritis, antiphospholipid syndrome, CREST, progressive systemic sclerosis, mixed tive tissue disease (Sharp syndrome), Reiter's syndrome, juvenile arthritis, cold agglutinin disease, essential mixed cryoglobulinemia, rheumatic fever, ankylosing spondylitis, chronic polyarthritis, myasthenia gravis, multiple sclerosis, chronic inflammatory inating polyneuropathy, Guillan-Barre syndrome, dermatomyositis/ polymyositis, autoimmune hemolytic anemia, thrompocytopenic a, penia, type I diabetes mellitus, ditis (including Hashimoto's and Grave'disease), Addison's disease, polyglandular syndrome, pemphigus (vulgaris, foliaceus, sebaceous and vegetans), bullous and cicatricial pemphigoid, pemphigoid gestationis, epidermolysis bullosa ita, linear IgA disease, lichen sclerosus et atrophicus, morbus Duhring, psoriasis vulgaris, e, generalized pustular and localized pustular sis, vitiligo, alopecia areata, primary biliary cirrhosis, autoimmune hepatitis, all forms of ulonephritis, pulmonal hemorrhage (goodpasture syndrome), IgA nephropathy, pernicious anemia and autoimmune gastritis, inflammatory bowel diseases (including s ulcerosa and morbus Crohn), Behcet's disease, Celic- Sprue disease, autoimmune uveitis, autoimmune myocarditis, granulomatous orchitis, atogenesis without orchitis, idiopatic and secondary ary fibrosis, inflammatory diseases with a possibility of autoimmune pathogensesis, such as pyoderma gangrensosum, lichen ruber, sarcoidosis (including Lofgren and ous/subcutaneous type), granuloma anulare, allergic type I and type IV immunolgical reaction, asthma bronchiale, osis, atopic, t and airborne dermatitis, large vessel vasculitis (giant cell and Takayasu's arteritis), medium sized vessel vasculitis (polyarteritis nodosa, Kawasaki disease), small vessel vasculitis (Wegener's granulomatosis, Churg Strauss syndrome, microscopic polangiitis, HenochSchoenlein purpura, essential cryoglobulinemic vasculitis, cutaneous leukoklastic angiitis), hypersensitivity syndromes, toxic mal necrolysis (Stevens-Johnson syndrome, erythema multiforme), diseases due to drug side effects, all forms of cutaneous, organ- specific and systemic effects due to type l-vu (Coombs classification) immunologic forms of reaction, transplantation related ogies, such as acute and chronic graft versus host and host versus graft disease, ing all organs (skin, heart, kidney, bone marrow, eye, liver, spleen, lung, muscle, central and peripheral nerve system, connective tissue, bone, blood and lymphatic vessel, genito-urinary system, ear, cartillage, primary and ary lymphatic system including bone marrow, lymph node, thymus, gastrointestinal tract, including oro-pharynx, esophageus, h, small intestine, colon, and rectum, including parts of above mentioned organs down to single cell level and substructures, e. g. stem cells).

Particularly ably, the disease is a neoplastic or autoimmune disease.

In an especially preferred embodiment the disease is cancer.

Examples of cancers in terms of the organs and parts of the body affected include, but are not limited to, the breast, , ovaries, colon, rectum, (including colon and rectum i.e. colorectal cancer), lung, (including small cell lung cancer, nonsmall cell lung cancer, large cell lung cancer and mesothelioma), bone, endocrine , adrenal gland, thymus, liver, stomach, intestine, (including gastric cancer), pancreas, bone marrow, hematological malignancies, (such as lymphoma, leukemia, myeloma or lymphoid malignancies), bladder, urinary tract, kidneys, skin, thyroid, brain, head, neck, prostate and . Preferably the cancer is selected from the group consisting of breast cancer, prostate cancer, cervical cancer, ovarian cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, brain , neuroendocrine cancer, lung cancer, kidney cancer, hematological malignancies, melanoma, T-cell leukemia and sarcomas. More especially ably the cancer is selected from the group consisting of breast cancer, cervical cancer, ovarian cancer, T-cell ia and lung cancer. In an especially preferred embodiment the cancer is selected from the group consisting of lung cancer, ovarian cancer and T-cell leukemia.

Samples The measurement of the level of ated n may be performed in vitro , on a sample of biological tissue derived from the subject. The sample may be any biological material separated from the body such as, for example, normal tissue, tumour tissue, cell lines, whole blood, serum, plasma, cerebrospinal fluid, lymph fluid, circulating tumour cells, cell lysate, tissue , urine and aspirates. ably the sample is derived from normal tissue, tumour tissue, or circulating tumour cells. More ably the sample is derived from tumour tissue or circulating tumour cells. In one particularly preferred embodiment the sample is derived from tumour tissue. For example, the level of acetylated tubulin may be measured in a fresh, frozen or formalin fixed/paraffin embedded tumour tissue sample.

The sample is pre-obtained from the subject before the sample is subjected to the method steps involving measuring the level of the biomarker. The methods for removal of the sample are well known in the art, and it may for example be removed from the t by biopsy, for example by punch biopsy, core biopsy or aspiration fine needle biopsy, endoscopic biopsy, or surface biopsy. Blood may be collected by venipuncture and r processed according to standard ques. Circulating tumour cells may also be ed from blood based on, for example, size (e.g. ISET - Isolation by Size of Epithelial Tumour cells) or magnetic cell enrichment. (e.g. Cellsearch®, Veridex, Raritan, NJ).

Sample comparison The subject may be human or animal. Preferably the subject is human.

The biomarker acetylated tubulin is measured ex vivo in a sample or samples taken from the human or animal body, preferably taken from the human body. The sample or samples are pre-obtained from the human or animal body, ably pre-obtained from the human body before the sample is subjected to the method steps involving measuring the level of the biomarker.

A biomarker is in general a substance that is used as an indicator of a biological se, preferably as an indicator of the susceptibility to a given treatment, which in the present application is treatment with a compound of general a I or a pharmaceutically acceptable derivative thereof.

In a particularly preferred embodiment, higher acetylated tubulin levels in the sample relative to a standard value or set of standard values predicts resistance.

As used , an increase or relatively high or high or higher levels relative to a standard level or set of standard levels means the amount or concentration of the ker in a sample is detectably greater in the sample relative to the standard level or set of standard levels. This encompasses at least an increase of, or higher level of, about 1% relative to the standard, preferably at least an increase of about % relative to the standard. More preferably it is an increase of, or higher level of, at least about 10% relative to the rd. More particularly preferably it is an increase of, or higher level of, at least about 20% ve to the standard. For example, such an increase of, or higher level of, may include, but is not limited to, at least about 1%, about 10%, about 20%, about 30%, about 50%, about 70%, about 80%, about 100%, about 150% or about 200% or more relative to the rd.

Preferably, higher acetylated n levels in a sample or samples i) relative to a standard value or set of standard values from subjects with the same tumour histotype; or ii) taken after treatment initiation and compared to a sample or samples taken from the same t before treatment initiation; or iii) relative to a standard value or set of standard values from normal cells or tissue; are predictive of resistance.

More preferably, higher acetylated tubulin levels in a sample or samples i) relative to a standard value or set of standard values from subjects with the same tumour histotype; or ii) taken after treatment initiation and compared to a sample or samples taken from the same subject before treatment initiation; are predictive of resistance.

Especially preferably, higher acetylated tubulin levels in a sample or samples taken after treatment initiation and compared to a sample or samples taken from the same subject before treatment initiation are predictive of resistance.

Also preferably, higher acetylated n levels in a sample or samples ve to a standard value or set of standard values taken from subjects with the same tumour histotype are predictive of resistance.

In one preferred embodiment, for the case i) where the measurement is compared in a sample or samples relative to a standard value or set of standard values taken from samples from subjects with the same tumour histotype as the sample to which it is to be compared, the standard value or set of standard values are established from samples taken from a population of subjects with that cancer type. The samples from these standard subjects may for example be d from the tumour tissue or blood, as long as the origin of the sample is tent n the standard and the sample to be compared.

In another preferred ment, for the case ii) where the measurement is compared in a sample or samples taken after treatment initiation and compared to a sample or s taken from the same subject before treatment initiation, it is measured preferably to predict acquired resistance. The samples are compared to cells or tissue from the same ical . The prediction of acquired resistance would then indicate that the treatment with the compound should be discontinued.

The biomarker is thus used to monitor whether further treatment with the compound is likely to give the required response (e.g. reduction of abnormal cells), or whether the cells have become non-responsive or ant to such ent.

In yet another preferred embodiment, for the case iii) where the measurement is compared in a sample or samples relative to a standard value or set of standard values taken from normal cells or tissue, the standard value or set of standard values may be established from a sample of normal (e.g. non-tumorous) cells or tissue or body fluid. Such data may be gathered from a population of subjects in order to develop the standard value or set of standard values.

The standard value or set of standard values are established ex-vivo from pre-obtained samples which may be from cell or preferably biological al taken from at least one subject and more preferably from an average of subjects (e.g., n=2 to 1000 or more). The standard value or set of standard values may then be correlated with the response data of the same cell lines, or same subjects, to treatment with a compound of general formula I or a pharmaceutically acceptable derivative thereof. From this correlation a comparator module, for example in the form of a relative scale or g system, optionally including cut-off or threshold values, can be established which indicates the levels of biomarker associated with a spectrum of response levels to the compound of formula I or a pharmaceutically able derivative thereof. The spectrum of response levels may comprise relative sensitivity to the eutic activity of the compound, (e.g. high ivity to low sensitivity), as well as resistance to the therapeutic activity. In a preferred ment this comparator module comprises a cut-off value or set of values which predicts resistance to treatment.

For e, if an immunohistochemical method is used to measure the level of acetylated tubulin in a sample, standard values may be in the form of a scoring system. Such a system might take into account the percentage of cells in which staining for acetylated tubulin is present. The system may also take into account the relative intensity of staining in the individual cells. The standard values or set of standard values of the level of acetylated tubulin may then be correlated with data indicating the se, ally resistance, of the subject or tissue or cell line to the therapeutic activity of a compound of formula I or a pharmaceutically acceptable derivative thereof. Such data may then form part of a comparator module.

Response is the reaction of the cell lines, or preferably of the subject, or more preferably of the disease in a subject, to the therapeutic activity of a nd of general formula I or a pharmaceutically able derivative thereof. The spectrum of response levels may se relative sensitivity to the therapeutic activity of the nd, (e.g. high sensitivity to low sensitivity), as well as resistance to the therapeutic activity. The response data may for example be monitored in terms of: objective response rates, time to disease progression, progression free al, and overall survival.

The response of a cancerous disease may be evaluated by using criteria well known to a person in the field of cancer treatment, for example but not restricted Response Evaluation Criteria in Solid Tumors T) Guidelines, Source: Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J. New se evaluation criteria in solid tumours: d RECIST ine (version 1.1). Eur J Cancer. 2009 ;45:228-47; RANO Criteria for High-Grade Gliomas, Source: Wen PY, Macdonald DR, Reardon DA, Cloughesy TF, Sorensen AG, Galanis E, Degroot J, Wick W, t MR, Lassman AB, Tsien C, Mikkelsen T, Wong ET, Chamberlain MC, Stupp R, Lamborn KR, Vogelbaum MA, van den Bent MJ, Chang SM. Updated response assessment criteria for high-grade gliomas: response assessment in neuro-oncology working group. J Clin Oncol. 8(11):1963-72; CA-125 Rustin ia for Ovarian Cancer Response, Source: Rustin GJ, Quinn M, Thigpen T, du Bois A, Pujade-Lauraine E, Jakobsen A, auer E, Sagae S, Greven K, Vergote I, tes A, Vermorken J. Re: New guidelines to evaluate the response to treatment in solid tumors (ovarian cancer). J Natl Cancer Inst. 2004; 96(6):487-8; PSA Working Group 2 Criteria for Prostate Cancer Response, Source: Scher HI, Halabi S, Tannock I, Morris M, Sternberg CN, Carducci MA, Eisenberger MA, Higano C, Bubley GJ, Dreicer R, Petrylak D, Kantoff P, Basch E, Kelly WK, Figg WD, Small EJ, Beer TM, Wilding G, Martin A, Hussain M; Prostate Cancer Clinical Trials Working Group. Design and end points of clinical trials for patients with progressive prostate cancer and te levels of testosterone: recommendations of the Prostate Cancer Clinical Trials Working Group. J Clin Oncol. 2008;26(7):1148-59.

Resistance is associated with there not being an observable and/or able ion in, or absence of, one or more of the following: reduction in the number of abnormal cells, preferably ous cells; or e of the abnormal cells, ably cancerous cells; for cancerous diseases: reduction in tumour size; inhibition (i.e., slowed to some extent and preferably stopped) of further tumour growth; reduction in the levels of tumour markers such as PSA and CA-125; inhibition (i.e., slowed to some extent and preferably d) of cancer cell infiltration into other organs (including the spread of cancer into soft tissue and bone); inhibition (i.e., slowed to some extent and preferably stopped) of tumour metastasis; ation of one or more of the symptoms associated with the specific cancer; and reduced morbidity and mortality.

In a preferred embodiment resistance means there is no observable and/or measurable reduction in, or absence of, one or more of the following criteria: reduction in tumour size; inhibition of r tumour growth, tion of cancer cell infiltration into other organs; and inhibition of tumour metastasis.

In a more preferred embodiment resistance refers to one or more of the following criteria: no ion in tumour size; no inhibition of further tumour growth, no inhibition of cancer cell infiltration into other organs; and no inhibition of tumour metastasis.

Measurement of the aforementioned resistance criteria is according to clinical guidelines well known to a person in the field of cancer treatment, such as those listed above for measuring the response of a cancerous disease.

Response may also be established in vitro by assessing cell proliferation and/or cell death. For example, effects on cell death or proliferation may be assessed in vitro by one or more of the following well established assays: A) Nuclear ng with Hoechst 33342 dye providing information about nuclear morphology and DNA fragmentation which are hallmarks of apoptosis. B) Annexin V binding assay which reflects the phosphatidylserine content of the outer lipid bilayer of the plasma membrane. This event is considered an early hallmark of apoptosis. C) TUNEL assay (Terminal deoxynucleotidyl transferase mediated dUTP Nick End ng assay), a fluorescence method for evaluating cells undergoing apoptosis or necrosis by measuring DNA fragmentation by labeling the al end of nucleic acids. D) FACs analysis of scently labeled cells (e.g. GFP [green fluorescence protein]- expressing cells). Dying or dead cells show a decreased fluorescence signal in comparison to living cells, allowing the quantification of induction of cell death. E) MTS proliferation assay measuring the metabolic activity of cells. Viable cells are metabolically active whereas cells with a compromised respiratory chain show a reduced activity in this test. F) Crystal violet staining assay, where s on cell number are monitored h direct staining of cellular components. G) eration assay monitoring DNA synthesis through incorporation of bromodeoxyuridine (BrdU).

Inhibitory effects on growth/proliferation can be directly determined. H) YO-PRO assay which involves a membrane impermeable, fluorescent, monomeric e, nucleic acid stain, which s analysis of dying (e.g. apoptotic) cells without interfering with cell viability. Overall effects on cell number can also be ed after cell permeabilisation. I) Propidium iodide staining for cell cycle distribution which shows alterations in distribution among the different phases of the cell cycle. Cell cycle arresting points can be determined. J) age-independent growth assays, such as colony outgrowth assays which assess the ability of single cell suspensions to grow into colonies in soft agar.

In a preferred embodiment ng to determination of resistance in vitro, resistance means there is no se in the proliferation rate of abnormal cells and/or reduction in the number of abnormal cells. More preferably resistance means there is no decrease in the proliferation rate of cancerous cells and/or no reduction in the number of cancerous cells. The reduction in the number of abnormal, preferably cancerous, cells may occur through a variety of programmed and non-programmed cell death mechanisms. Apoptosis, e-independent programmed cell death and autophagic cell death are examples of programmed cell death. However the cell death criteria involved in embodiments of the invention are not to be taken as limited to any one cell death mechanism.

Acetylated tubulin Preferred examples of the protein sequence of alpha and beta n (human alpha and beta tubulin) are listed in SEQ. ID No. 1-6, Figures 9-14. Alpha or beta tubulin, especially alpha tubulin, is a precursor of ated tubulin. As bed previously, a ic lysine e in the tubulin chain may be acetylated or deacetylated. The term acetylated tubulin also encompasses homologues, mutant forms, allelic variants, isoforms, splice variants and equivalents of the sequences represented by SEQ ID NO 1-6, with the proviso that a lysine e in the sequence is acetylated. More preferably it encompasses sequences having at least about 75% identity, especially preferably at least about 85% identity, particularly preferably at least about 95% identity, and more particularly preferably about 99% ty to said sequences, with in each case the proviso that a lysine residue in the sequence is acetylated. Particularly preferably lysine 40 of alpha tubulin is acetylated.

Level of acetylated tubulin The level of acetylated tubulin may be assayed in the sample by protein analysis techniques well known to a skilled person. Examples of methods known in the art which are suitable to measure the level of acetylated tubulin at the protein level include, but are not limited to, i) immunohistochemistry (IHC) analysis, ii) western blotting iii) immunoprecipitation iv) enzyme linked immunosorbant assay (ELISA) v) radioimmunoassay vi) Fluorescence activated cell sorting (FACS) vii) mass spectrometry, including matrix assisted laser desorption/ionisation (MALDI, e.g.

MALDI-TOF) and electrospray ionisation pectrometry (ESI—MS).

The antibodies involved in some of the above methods may be onal or polyclonal antibodies, antibody fragments, and/or various types of synthetic dies, including chimeric antibodies. The antibody may be labeled to enable it to be detected or capable of detection following reaction with one or more r species, for example using a secondary antibody that is labeled or capable of producing a detectable . Antibodies specific to the acetylated alpha tubulin are available commercially from Sigma. Additionally antibodies to acetylated alpha tubulin and acetylated beta tubulin can be ed via conventional dy generation methods well known to a skilled person.

Preferred methods of protein analysis are ELISA, mass spectrometry techniques, immunohistochemistry and western blotting, more preferably western blotting and immunohistochemistry. In n blotting, also known as immunoblotting, labelled antibodies may be used to assess levels of protein, where the intensity of the signal from the detectable label corresponds to the amount of protein, and can be quantified for example by densitometry.

Immunohistochemistry again uses labelled antibodies to detect the presence and relative amount of the biomarker. It can be used to assess the percentage of cells for which the biomarker is present. It can also be used to assess the localisation or relative amount of the biomarker in individual cells, the latter is seen as a function of the intensity of staining.

ELISA stands for enzyme linked immunosorbant assay, since it uses an enzyme linked to an dy or antigen for the detection of a specific protein. ELISA is lly performed as follows (although other ions in methodology exist): a solid substrate such as a 96 well plate is coated with a primary dy, which recognises the biomarker. The bound biomarker is then recognised by a secondary dy specific for the biomarker. This may be directly joined to an enzyme or a third anti-immunoglobulin antibody may be used which is joined to an enzyme. A substrate is added and the enzyme catalyses a reaction, yielding a specific .

By ing the optical density of this colour, the presence and amount of the biomarker can be determined.

Uses of ker The biomarker may be used to t acquired resistance of the disease in a subject to the compound of general formula I or a pharmaceutically acceptable derivative thereof as defined above.

The biomarker may be used to select subjects suffering or predisposed to suffering from a disease, preferably cancer, for treatment with a compound of general formula I or a pharmaceutically acceptable derivative thereof as defined above. The levels of such a biomarker may be used to identify subjects likely to d or to not respond or to ue to respond or to not continue to respond to treatment with such agents. Preferably the levels of such a biomarker may be used to identify ts likely to continue to respond or to not continue to respond to treatment with such agents. Stratification of subjects may be made in order to avoid unnecessary treatment regimes. In particular the biomarker may be used to identify subjects from whom a sample or samples do not display a higher level of acetylated tubulin, relative to a standard level or set of rd levels, whereupon such subjects may then be selected for treatment with the compound of formula I or a pharmaceutically acceptable derivative thereof as defined above. More particularly the ker may be used to identify subjects from whom a sample or samples do not display a higher level of acetylated tubulin, relative to a level ed before treatment tion, whereupon such ts may then be selected for continued treatment with the compound of formula I or a pharmaceutically acceptable derivative thereof as defined above.

The biomarker may also be used to assist in the determination of treatment regimes, regarding amounts and schedules of . onally, the biomarker may be used to assist in the selection of a combination of drugs to be given to a subject, ing a compound or compounds of l formula I or a pharmaceutically acceptable derivative thereof, and another chemotherapeutic (cytotoxic) agent or agents. Furthermore, the biomarker may be used to assist in the determination of therapy strategies in a subject including whether a compound of general formula I or a pharmaceutically acceptable derivative thereof is to be administered in combination with targeted therapy, endocrine therapy, radiotherapy, immunotherapy or surgical ention, or a combination of these.

Acetylated tubulin may also be used in combination with other biomarkers to predict the response to a compound of general formula I or a pharmaceutically acceptable tive thereof and to ine treatment regimes. It may furthermore be used in combination with chemo-sensitivity testing to predict resistance and to determine treatment regimes. Chemo-sensitivity testing involves directly applying a compound of general formula I to cells taken from the t, for example from a subject with haematological ancies or accessible solid tumours, for example breast, head and neck cancers or melanomas, to determine the response of the cells to the compound.

Method of treatment Also described is a method of treatment and acetylated tubulin for use in a method of treatment, wherein the level of acetylated tubulin is first established relative to a standard level or set of standard levels or pre-treatment initiation levels and then a compound of general formula I or a pharmaceutically acceptable derivative thereof as defined above, is administered if the level of acetylated tubulin in said sample is not higher than standard value or set of standard values or has not increased relative to pre-treatment initiation levels respectively. The compound of a I or a ceutically acceptable derivative f may be administered in a pharmaceutical composition, as is well known to a person d in the art. Suitable compositions and dosages are for example sed in A1 pages 35-39, which are specifically incorporated by reference herein. Compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as intravenous, intramuscular or subcutaneous administration, to looded animals, especially humans, are especially preferred. More particularly, compositions for intravenous administration are preferred.

The compositions comprise the active ingredient and a pharmaceutically acceptable carrier. An example of a composition includes, but is not limited to, the following: 5000 soft n capsules, each sing as active ingredient 0.05 g of one of the compounds of general formula (I), are prepared as follows: 250 g pulverized active ingredient is suspended in 2 liter Lauroglykol (propylene glycol laurate, ossé S.A., Saint Priest, France) and ground in a wet pulverizer to produce a particle size of about 1 to 3 µm. 0.419 g portions of the e are then introduced into soft gelatin capsules using a capsule-filling e.

Also described is a method of ng a neoplastic or autoimmune disease, preferably cancer, by first decreasing the level of acetylated tubulin in a subject that has a sample with a higher level of acetylated n compared to a standard level or set of standard levels or eatment initiation levels, and then treating the subject with a compound of general formula I or a pharmaceutically acceptable derivative as defined above. The level of acetylated tubulin may be decreased by direct or indirect chemical or genetic means. es of such methods are treatment with a drug that results in reduced acetylated tubulin expression, targeted ry of viral, plasmid or peptide constructs, or antibody or siRNA or antisense to gulate the level of acetylated tubulin. For example siRNA may be used to reduce the level of alphaTAT1 or delivery of a plasmid may be used to increase the expression of SirT2, and thereby reduce the level of acetylated tubulin in the cell. The subject may then be treated with a compound of general formula I or a pharmaceutically acceptable derivative thereof.

A compound of general a I or a pharmaceutically acceptable derivative thereof can be stered alone or in combination with one or more other eutic agents. Possible combination therapy may take the form of fixed combinations, or the administration of a compound as described herein and one or more other therapeutic agents which are staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic agents. A compound of general formula I or a pharmaceutically acceptable derivative thereof can, s or in addition, be administered especially for tumour therapy in combination with chemotherapy (cytotoxic therapy), targeted therapy, endocrine therapy, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term y is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient's status after tumour regression, or even chemo-preventive therapy, for example in ts at risk.

Kit and device Also bed is a kit and in another aspect to a device for predicting the response, preferably of a disease in a subject, to a compound of general a I or a pharmaceutically acceptable derivative thereof as defined above, comprising reagents necessary for measuring the level of acetylated tubulin in a sample.

Preferably, the reagents comprise a capture t sing a detector for ated tubulin and a detector reagent.

The kit and device may also preferably comprise a comparator module which comprises a standard value or set of standard values to which the level of acetylated tubulin in the sample is compared. In a preferred embodiment, the comparator module is included in instructions for use of the kit. In another red embodiment the comparator module is in the form of a display device, for example a strip of colour or numerically coded material which is designed to be placed next to the readout of the sample measurement to indicate ance levels. The standard value or set of standard values may be determined as described above.

The reagents are preferably antibodies or antibody fragments which selectively bind to ated tubulin. These may for e be in the form of one specific primary dy which binds to acetylated tubulin and a secondary antibody which binds to the primary antibody, and which is itself labelled for detection.

Alternatively, the primary antibody may also be labelled for direct detection. The kits or devices may optionally also contain a wash solution(s) that selectively allows retention of the bound biomarker to the e reagent as compared with other biomarkers after washing. Such kits can then be used in ELISA, western blotting, flow cytometry, immunohistochemistry or other immunochemical methods to detect the level of the biomarker.

More preferably the kit comprises a compound of general formula I, or a pharmaceutically acceptable derivative thereof as defined above. This compound may then be administered to the subject, in accordance with the level of the biomarker in the sample from the subject, as measured by the reagents comprised in the kit. Therefore the kit according to the invention may be used in the method of treatment, as defined above. In an especially preferred embodiment the kit ses a compound of the ing formula or a pharmaceutically able salt thereof N N N N O In a particularly preferred embodiment of the kit the pharmaceutically acceptable salt is a ochloride salt. Also described is the use of such a kit as described above.

Furthermore the device may comprise imaging devices or measurement devices (for e, but not restricted to, measurement of scence) which further process the measured signals and transfer them into a scale in a comparator module.

In the present specification the words “comprise” or “comprises” or “comprising” are to be understood as to imply the inclusion of a stated item or group of items, but not the exclusion of any other item or group of items.

Experimental methodology fluorescent staining of cultured cells A549 human non-small cell lung cancer (NSCLC, ATCC reference number CCL-185) cells, HeLa cervical cancer cells (ATCC reference number CCL-2) and SKBR3 breast carcinoma cells (ATCC reference number HTB-30) were seeded at ies of 50% on round microscope coverslips and cultured for 24 hours in RPMI- 1640 containing 10 % FCS (also referred to as FBS) at 37°C, 5% CO2. Compounds to be tested were dissolved in DMSO. The cell culture medium was replaced with medium containing the diluted compound(s) taxel, vinblastine, colchicine and nocodazole were purchased from Sigma-Aldrich) or e. After treatment for the times indicated in the Brief Description of the s, coverslips were washed and cells were fixed in methanol/acetone (1:1) for 5 minutes at room temperature and subsequently incubated in blocking buffer (0.5% BSA and 0.1% TX-100 in PBS) for minutes at room temperature. Specimens were then incubated with anti-alpha tubulin antibody , 1:2000) for 1 hour at room temperature in blocking buffer.

After l washing steps cells were incubated with AlexaFluor-488 goat-antimouse IgG (Molecular Probes, 1:3000) for 1 hour at room temperature followed by several washing steps with blocking buffer. Specimens were then d with ProLong Gold antifade (Molecular ) sealed with nail polish and examined with a Leica immunofluorescence microscope. Images were ed with a cooled CCD- camera and processed by ImageJ software.

Generation and Crystal Violet or Cell Death Assay of BAL27862-Resistant Cell Lines BAL27862-resistant sublines of human non-small cell lung cancer (H460 ATCC reference HTB-177; A549 ATCC reference CCL-185), ovarian cancer (SKOV3 ATCC reference HTB-77) and T-cell leukemia (Jurkat ATCC reference 2) cell lines were generated by long-term selection in complete cell culture medium (RPMI- 1640 containing 10 % FBS; Sigma-Aldrich) by increasing BAL27862 concentrations in a stepwise fashion. Dependent on the cell line, the ion process was carried out for 8-12 months in order to achieve resistance factors (ratio of IC50 or EC50 of resistant cell line versus the IC50 or EC50 of the appropriate parental cell line) between 3 and 22.5. The ance factors were determined for the adherent cell lines H460, A549 and SKOV3 by measuring proliferation using the Crystal Violet assay, whereas for the suspension Jurkat cell line cell death was measured using FACS. The resistant sub lines were ed at the highest tolerated BAL27862 concentration and subsequently frozen and stored in liquid nitrogen.

A549 (2000 cells/well)cells/well) , H460 (1000 cells/well) and SKOV3 /well) and SKOV3 (2000 cells/well) cells were seeded in 96 well platesells were seeded in 96 well plates. After 24 hours incubation, the cellsthe cells were incubated for 72 hours with DMSO, BAL27862, colchicine, zole, paclitaxel or vinblastine diluted in complete medium (final concentration DMSO max. 0.5 %). After medium was removed, cells were fixed and stained by adding 50 µlmedium was removed, cells were fixed and stained by adding 50 µl Crystal Violet stain (0.2 % Crystal Violet in 50 % Methanol) per well. Plates were incubated for 1(0.2 % Crystal Violet in 50 % Methanol) per well. Plates were incubated for 1 hour at room temperature. uently the stain was decanted and plates were washed 4 times with double-distilled water. Plates were air-dried for several hours.washed 4 times with double dried for several hours.

Stain was dissolved by adding 100 µl buffer (0.1 M Tris pH 7.5, 0.2 % SDS, 20 %olved by adding 100 µl buffer (0.1 M Tris pH 7.5, 0.2 % SDS, 20 % Ethanol) per well and g the plates. ance at 590 nm was measured using a SpectraMax M2e plate reader (Molecular Devices). using a SpectraMax M2e plate reader (Molecular Devices).

Jurkat cells stably expressing GFP (green fluorescence pJurkat cells stably expressing GFP (green fluorescence protein) wererotein) were incubated with increasing BAL27862BAL27862 concentrations in culture medium for 72 hconcentrations in e medium for 72 hours and then analyzed by FACS (excitation at 488 nM, emission at 525 nM). Dying orand then analyzed by FACS (excitation at 488 nM, emission at 525 nM). dead cells, showing a decreased fluorescence activity in comparison to living cellsshowing a decreased fluorescence ty in comparison to living cellsshowing a decreased fluorescence activity in comparison to living cells, were quantified (Green fluorescent protein as a novel tool to measure apoptosis en fluorescent protein as a novel tool to measure sis and necrosis . Strebel et al, 20012001 , Cytometry 43(2): 126-133).

Antiproliferative IC5050 and cell death EC50 values were ated fromalues were calculated from concentration response curves using GraphPad Prism software. Resistance factors were ated as a ratio ofwere calculated as a ratio of the IC50 or EC50 in the resistant line tin the resistant line variant versus the IC 50 or EC50 in the parental line.in the parental line.

Protein Analysis Cells were washed with e washed with ice-cold PBS containing 1 mM phenylmethylsulfonylmM phenylmethylsulfonyl fluoride (PMSF) and with icefluoride (PMSF) and with ice-cold buffer containing 50 mM HEPES (pH 7.5), 150mM HEPES (pH 7.5), 150 mM NaCl, 25 mM β-glycerophosphate, 25glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 15 mMmM EDTA, 15 mM pyrophosphate, 2 mM sodiummM sodium orthovanadate, 10 mM sodium molybdate,mM sodium molybdate, leupeptin (10 µg/mL), aprotinin (10 µg/mL) and 1µg/mL) and 1 mM PMSF. Cells were extracted in the samemM PMSF. Cells were extracted in the same buffer ning 1 % NP40. After homogenization, lysates were clarified bybuffer containing 1 % NP40. After homogenization, lysates were clarified by centrifugation and frozen atand frozen at 80°C.

Protein concentration was determined with the BCA Protein Assay e).

Immunoblotting was performed using 20 µg of total protein per lane. Protein was separated on a 10 % SDS-gel and transferred to a PVDF membrane using y Blotting (90 min, 50 mA/gel). Tubulin ation was measured using a mouse monoclonal anti-acetylated tubulin antibody (Sigma, reference number T7451): 1:10’000 dilution in PBS 5 % milk / 0.1 % Tween. The actin antibody used was a mouse monoclonal (Chemicon, reference number MAB1501): 1:5’000 dilution in PBS % milk / 0.1 % Tween. The secondary dy used for immunoblotting was peroxidase-conjugated goat anti-mouse on ImmunoResearch Laboratories INC: # 115146 JIR), used at a dilution of 1:5’000 in PBS plus 0.5 % milk and 0.1 % Tween. Labelled bands were revealed using a Raytest Stella 3200 High Performance g System.

Detailed examples Example 1: A distinct mitotic phenotype induced by compounds of general a I Treatment with compound A (BAL27862) or with compound B or compound C, induced a highly reproducible and distinct microtubule phenotype in all tumour cell lines tested (shown for compound A in A549, HeLa and SKBR3 cells in Figure 1, and for compound B and nd C in A549 cells in Figure 2). In dividing cells an apparent fragmentation of the mitotic spindle occurred, resulting in the formation of dot-like structures (Figure 1). This phenotype was shown to be distinct from that observed with conventional microtubule targeting agents, such as the microtubule stabiliser paclitaxel and the microtubule destabilisers stine and cine (Figure 3) and nocodazole (Figure 4).

Example 2: BAL27862 overcomes microtubule phenotype induced by conventional microtubule-targeting drugs in a dominant fashion In order to show the uniqueness of its ty on microtubules, BAL27862 was tested in combination with vinblastine, colchicine and paclitaxel (Figure 5) and nocodazole (Figure 6) using A549 cells. Treatment with vinblastine, colchicine, paclitaxel or nocodazole alone induced the mitotic microtubule phenotypes characteristic of these agents. However, combination treatment with BAL27862 for the last 4 hours resulted in disruption of the ubule structures; creating a phenotype consistent with treatment of BAL27862 alone, despite the continued presence of vinblastine, colchicine, paclitaxel or nocodazole. In contrast, treating first with BAL27862 and uently for 4 hours in combination with vinblastine, cine, paclitaxel or nocodazole had no impact on the observed microtubule phenotype that was tent with treatment with BAL27862.

These data demonstrate that compounds of formula I affect microtubule biology consistently, but in a different manner than conventional microtubule targeting .

Detailed Examples ing to the invention Example 3: Increased acetylated tubulin expression is observed in tumour lines selected for ance to a compound of general formula I In vitro selection for resistance to BAL27862 resulted in the generation of 4 relatively resistant tumour cell lines, with the following resistance factors versus al lines (based on IC50 [A549, SKOV3, H460] or EC50 [Jurkat] determinations using the Crystal Violet or FACs cell death assay, respectively): A549 (3.0 fold); SKOV3 resistant 1 (7.6 fold); SKOV3 resistant 2 (11.6 fold); H460 (5.3 fold); Jurkat (22.5 Fold) (Table 1).

Table 1: Resistance factors (ratio IC50 or EC50 62-resistant cell Treatment line variant versus IC50 or EC50 parental cell line) compound SKOV3 SKOV3 Jurkat A549 H460 resistant 1 resistant 2 BAL27862 3.0 5.3 7.6 11.6 22.5 Colchicine 0.9 1.6 2.0 2.8 nd Nocodazole 1.6 1.3 3.6 3.9 nd Vinblastine 2.3 4.6 15.7 17.8 nd Paclitaxel 0.06 0.3 0.4 0.5 nd nd: not ined In general these BAL27862-resistant cells exhibited a different level of response to other microtubule destabilising agents, such as colchicine, nocodazole and vinblastine, as compared to BAL27862; and indeed sed sensitivity to the microtubule stabiliser paclitaxel was observed in all lines tested (Table 1).

Extraction and immunoblot analysis of these lines to measure the acetylated n levels, ed by comparison to BAL27862 resistance data (Table 1), showed that ated tubulin is higher in the resistant lines, as compared to the parental lines (Figure 7). This was maintained throughout resistance development in the SKOV3 cells (Figure 8). These data show the association of sed acetylated tubulin expression levels with acquired resistance to BAL27862.

List of abbreviations A549 human non-small cell lung cancer cell line alphaTAT1 alpha-tubulin N-acetyltransferase 1 BCA bicinchoninic acid BrdU bromodeoxyuridine BSA bovine serum albumin CA-125 cancer antigen 125 CCD charge-coupled device cDNA complementary deoxyribonucleic acid CREST limited scleroderma syndrome DMSO dimethylsulphoxide DNA deoxyribonucleic acid dUTP 2´-Deoxyuridine 5´-Triphosphate EDTA Ethylenediaminetetraacetic acid EGTA Ethyleneglycol-bis (β-aminoethyl)-N,N,N ′,N ′-tetraacetic acid ELISA -linked immunosorbent assay Elp3 elongator x protein 3 ESI-MS electrospray ionisation mass-spectrometry FACS fluorescence activated cell scan/sorting FCS/FBS foetal calf / foetal bovine serum G2/M transition from G2 to the mitotic phase in the cell cycle GFP green fluorescence protein HDAC6 histone deacetylase 6 HeLa human squamous cell cancer cell line HEPES 4-(2-Hydroxyethyl)piperazineethanesulphonic acid IHC Immunohistochemistry ISET Isolation by size of epithelial tumor cells Jurkat human T-cell leukemia cell line MAB monoclonal dy MALDI matrix-assisted-laser-desorption/ionisation massspectrometry TOF matrix-assisted-laser-desorption/ionisation–time-of-flightmass-spectrometry MDR1 multi-drug resistant protein mRNA messenger ribonucleic acid MTS 3-(4,5-dimethylthiazolyl)(3-carboxymethoxyphenyl) (4-sulphophenyl)-2H-tetrazolium NCBI National center for Biotechnology Information NSCLC non-small cell lung cancer NP40 Nonidet P40 PBS ate buffered saline P-gp oprotein PMSF phenylmethylsulphonyl fluoride PSA prostate-specific antigen PVDF polyvinylidene fluoride RANO response assessment for high-grade gliomas RECIST response evaluation criteria in solid tumors RPMI-1640 cell culture medium used for culturing transformed and non-transformed eukaryotic cells and cell lines SDS sodium dodecyl sulphate SEQ. ID No. sequence identification number siRNA small inhibitory ribonucleic acid SirT2 NAD-dependent deacetylase sirtuin-2 SKBR3 human mammary carcinoma cell line SKOV3 human ovarian oma cell line TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling Tween rbate 20, non-ionic detergent TX-100 Triton-X100 YO-PRO fluorescent, monomeric cyanine, nucleic acid stain The term ‘comprising’ as used in this specification and claims means ‘consisting at least in part of’. When interpreting statements in this specification and claims which includes the ‘comprising’, other es besides the es prefaced by this term in each statement can also be present. Related terms such as ‘comprise’ and ‘comprised’ are to be interpreted in similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other s of information, this is generally for the purpose of providing a context for sing the features of the invention. Unless specifically stated otherwise, reference to such external nts is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

Claims (28)

We claim:

1. Use of acetylated tubulin as a biomarker for ting the response to a compound, n the compound is a compound of general formula I R3 R2 N R4 N R5 N N 5 wherein R represents phenyl, thienyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, 10 y-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted amino wherein the two substituents on nitrogen form together with the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, lower 15 alkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacent tuents are methylenedioxy; and wherein pyridinyl is optionally substituted by lower alkoxy, amino or halogen; X represents a group C=Y, wherein Y stands for oxygen or nitrogen tuted by 20 hydroxy or lower alkoxy; R1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or lower alkyl; R2, R3 and R6 represent hydrogen; 25 R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R4 and R5 together represent methylenedioxy; and pharmaceutically acceptable salts, solvates, esters and amides of naturally ing amino acids, small peptides or pegylated hydroxy acids, salts of such 5 esters and amides, and polymorphs thereof; or n R represents phenyl or pyridinyl wherein phenyl is optionally tuted by one or two substituents independently 10 selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower carbonylamino, lower arbonylamino, substituted amino wherein the two substituents on nitrogen form 15 er with the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, , cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein pyridinyl is optionally substituted by lower alkoxy, amino or halogen; 20 X represents ; R1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R2, R3 and R6 represent hydrogen; 25 R4 and R5, independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R4 and R5 together represent methylenedioxy; and pharmaceutically acceptable salts, solvates, esters and amides of naturally ing amino acids, small peptides or pegylated hydroxy acids, salts of 30 such esters and amides, and polymorphs thereof, and wherein the prefix lower denotes a radical having up to and including a maximum of 7 carbon atoms and wherein the response is of a disease in a subject and the biomarker acetylated tubulin is measured ex vivo in a sample or samples taken from the human or animal body.

2. Use according to claim 1, wherein in the compound of general formula I 5 R ents phenyl or pyridinyl; wherein phenyl is optionally substituted by one or two substituents ndently selected from lower alkyl, lower , amino, acetylamino, halogen and nitro; and wherein pyridinyl is ally substituted by amino or halogen; 10 X represents a group C=O; R1 represents hydrogen or cyano-lower alkyl; R2, R3, R4, R5 and R6 represent hydrogen; 15 and pharmaceutically acceptable salts, solvates, esters and amides of naturally occurring amino acids, small peptides or pegylated hydroxy acids, salts of such esters and amides, and polymorphs thereof, and wherein the prefix lower denotes a radical having up to and including a m 20 of 7 carbon atoms.

3. Use according to claim 1 or claim 2, wherein the compound is represented by the following formula N N wherein R, Y and R1 are defined as follows: R Y R1 O CH2CH2CN O H O CH2CH2CN NH N or pharmaceutically acceptable salts, solvates, esters and amides of naturally ing amino acids, small peptides or pegylated hydroxy acids, salts of such 5 esters and amides, and polymorphs thereof.

4. Use according to any one of claims 1 to 3, wherein the compound is N N N N O or pharmaceutically acceptable salts, solvates, esters and amides of naturally occurring amino acids, small peptides or pegylated y acids, salts of 10 such esters and amides, and polymorphs thereof.

5. Use according to any one of claims 1 to 4, wherein the prefix lower denotes a l having up to and including a maximum of 4 carbon atoms.

6. Use ing to any one of claims 1 to 5, wherein an amide of the compound of general formula I is used as a prodrug, the amide being formed from an amino group t within the R group and the carboxy group of glycine, alanine or lysine. 5

7. Use according to any one of claims 1 to 6, wherein the compound is N N N N O or a pharmaceutically acceptable salt thereof.

8. Use ing to claim 7, wherein the pharmaceutically able salt is a hydrochloride salt or dihydrochloride salt.

9. Use ing to any one of claims 1 to 8, for predicting the resistance of the disease in the subject to said compound. 15

10. Use according to any one of claims 1 to 9, wherein the disease is a neoplastic disease or autoimmune disease.

11. Use according to claim 10, wherein the disease is cancer.

12. Use according to any one of claims 1 to 11, wherein the disease is selected from the group consisting of breast cancer, prostate cancer, cervical cancer, ovarian cancer, gastric cancer, colorectal , pancreatic cancer, liver cancer, brain cancer, neuroendocrine cancer, lung cancer, kidney , hematological malignancies, melanoma, T-cell leukemia, and sarcomas.

13. Use according to claim 12, wherein the disease is selected from the group 5 consisting of breast cancer, al cancer, ovarian cancer, T-cell leukemia and lung cancer.

14. Use according to claim 13, wherein the disease is selected from the group ting of lung cancer, ovarian cancer and T-cell leukemia.

15. Use according to any one of claims 1 to 14, n a higher level of 10 acetylated tubulin in the sample from the subject relative to a standard value or set of standard values levels or eatment initiation levels predicts resistance.

16. Use according to claim 15, wherein higher acetylated tubulin levels in a sample or samples i) relative to a standard value or set of rd values from subjects with 15 the same tumour histotype; or ii) taken after treatment initiation and compared to a sample or samples taken from the same subject before ent tion; or iii) relative to a standard value or set of standard values from normal cells or tissue; 20 are predictive of resistance.

17. Use according to any one of claims 1 to 16, wherein the biomarker is used to select subjects suffering or predisposed to suffering from a disease for treatment with a compound of general formula I or a pharmaceutically acceptable salt, solvate, ester or amide of a naturally occurring amino acid, small peptide or 25 pegylated hydroxy acid, or polymorph thereof as defined in any one of claims 1 to 8.

18. Use according to claim 17 wherein the disease is .

19. Use according to any one of claims 1 to 18, wherein the sample is derived from normal tissue, tumour tissue, or circulating tumour cells.

20. Use according to claim 19, wherein the sample is d from tumour 5 .

21. A method for predicting in a subject suffering from a cancer the response of that cancer to a compound of l formula I or a pharmaceutically able salt, solvate, ester or amide of a naturally occurring amino acid, small 10 peptide or pegylated hydroxy acid, or a salt of such ester or amide, or polymorph thereof as defined in any one of claims 1 to 8, comprising the steps of: a1) measuring ex vivo a level of acetylated tubulin in a sample pre-obtained from tumour tissue or circulating tumour cells of the subject suffering from the disease, which is a cancer, to obtain a value or values representing this level; or 15 a2) measuring ex vivo a level of acetylated tubulin in a sample pre-obtained from the subject suffering from the disease, after initiation of treatment with the compound of general formula I, to obtain a value or values representing this level; b1) comparing the value or values from step a1) to a rd value or set 20 of standard values from subjects with the same cancer type, wherein a higher level of acetylated tubulin in the sample from the subject relative to the standard value or set of standard values predicts resistance; or b2) comparing the value or values from step a2) to the pre-treatment initiation level of that subject, wherein a higher level of acetylated tubulin in the 25 sample after ent initiation relative to the pre-treatment initiation level predicts acquired resistance.

22. Use of a compound of general formula I or of a pharmaceutically acceptable salt, solvate, ester or amide of a naturally occurring amino acid, small peptide or ted hydroxy acid, or a salt of such ester or amide, or polymorph thereof as defined in any one of claims 1 to 8, for the preparation of a ceutical 5 composition for treating a cancer in a human t in need thereof, wherein the human subject is selected for treatment with the compound of general a I or with the salt, e, ester or amide of a naturally occurring amino acid, small e or pegylated hydroxy acid, or salt of such ester or amide, or polymorph thereof as defined in any one of claims 1 to 8, if the level of acetylated tubulin, 10 ed ex vivo in a sample taken from the human subject, is not higher than a standard value or set of rd values from subjects with the same tumour histotype or from normal cells, tissue or body fluid, wherein a higher value of acetylated tubulin in the sample obtained from the human subject relative to the standard value or set of standard values is predictive of resistance to the compound 15 of formula (I), or to the pharmaceutically acceptable salt, solvate, ester or amide of a naturally occurring amino acid, small e or pegylated hydroxy acid, or to the salt of such ester or amide, or to the polymorph thereof.

23. A kit when used for predicting the response to a compound of general formula I or a pharmaceutically acceptable salt, solvate, ester or amide of a naturally 20 occurring amino acid, small peptide or pegylated hydroxy acid, or a salt of such ester or amide, or polymorph thereof, as defined in any one of claims 1 to 8, comprising reagents necessary for measuring a level of acetylated tubulin in a sample taken from a subject with a cancer, and further comprising a comparator module which comprises a standard value or set of standard values of a level of acetylated tubulin 25 taken from samples of tumour tissue or circulating tumour cells of subjects with a cancer of the same histotype to which the level of acetylated tubulin in the sample is compared, wherein said reagents comprise a capture reagent comprising a detector for acetylated n as defined in claim 1 and a detector reagent, 30 and n the kit comprises a compound of the following formula or a pharmaceutically acceptable salt thereof, N N N N O

24. The kit according to claim 23, wherein the capture reagent is an antibody. 5

25. The kit according to claim 23, wherein the pharmaceutically acceptable salt is the dihydrochloride salt.

26. A use as claimed in any one of claims 1 to 20 or 22, substantially as herein described with reference to any example thereof.

27. A method as d in claim 21, ntially as herein described with 10 reference to any example thereof.

28. A kit as claimed in any one of claims 23 to 25, substantially as herein described with reference to any example thereof. WO 13802